Summary auto-generated
This study characterized the binding and membrane fusion of Autographa californica nucleopolyhedrovirus (AcMNPV) budded virus to cultured Spodoptera frugiperda (Sf21) insect cells. Using radioactively labeled and fluorescently labeled viruses, researchers determined that AcMNPV binds to specific cell surface receptors with approximately 3,100 binding sites per cell. Virus binding was influenced by pH, buffer composition, and cation concentrations, with optimal binding at intermediate cation levels. Protease treatments reduced binding, suggesting protein receptors are involved, while glycosidase treatments had no effect. Fluorescence microscopy and kinetic studies revealed that virus entry occurs primarily through endocytosis, with envelope-membrane fusion triggered by acidification in endocytic vesicles rather than at the plasma membrane. The study also tested whether a Trichoplusia ni granulovirus enhancin protein could enhance binding or fusion but found no significant effects. These findings support the established concept that baculoviruses enter cultured insect cells via an endocytic pathway rather than direct membrane fusion at the cell surface.
Key findings
- AcMNPV binds specifically to ~3,100 receptor sites per Sf21 cell with a dissociation constant of 4.3×10⁻¹¹ M
- Virus binding depends on pH, buffer composition, and divalent cation concentration, with optimal binding at 10 mM cation concentration
- Protease treatment reduces virus binding, indicating proteinaceous receptors; glycosidase treatment has no effect
- Membrane fusion is triggered by acidification within endocytic vesicles, confirming that endocytosis is the primary entry pathway
- TnGV enhancin has no measurable effect on AcMNPV binding or membrane fusion in cultured cells
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Abstract
Binding of baculoviruses to insect cells and fusion of the virus envelope to cell membranes are early events suggested to be affected by baculovirus enhancins. The binding of Autographa californica nucleopolyhedrovirus (AcMNPV) to the Spodoptera frugiperda cell line Sf21 and the fusion of the virus envelope to cell membranes were characterized. Virus binding assays demonstrated that AcMNPV budded virus (BV) bound to specific binding sites on Sf21 cells with an avidity of 2.3 x 10(10) M(-1). The cells displayed 3.1 x 10(3) specific binding sites per cell in a confluent monolayer. In addition, the effects of pH, buffer composition and cation concentration on the binding were examined. Using a fluorescent probe (R18) and fluorescence microscopy, the fusion of AcMNPV BV envelope to the cell membrane was directly visualized in living cells. It has been reported that Trichoplusia ni nucleopolyhedrovirus enters Sf21 cells by membrane fusion at the cell surface; however, the present studies confirmed the well established concept that adsorptive endocytosis is the major entry pathway for baculovirus BV infection. Membrane fusion kinetics and fluorescence microscopy demonstrated and verified that the envelope- cell membrane fusion was triggered by acidification. The effect of a T. ni granulovirus enhancin on virus binding and membrane fusion was examined, and no increase in activity was observed.